Rev and Tat are regulatory factors of the human immunodeficiency virus (HIV). They are among the first proteins to be synthesized in infected cells, and both are stimulators of HIV gene expression. The biological activity of Tat and Rev is important for the amount of virus production by infected cells. For example, the low HIV production which is characteristic for human astrocytes is associated with a 10 times decreased activity of HIV Rev (Ludwig et al., 1999, J. Virol. 73:8279–8289).
This importance mentioned above of Rev and Tat for the amount of virus production renders them particularly suitable for methods for the detection of HIV infection.
At present, there are two types of REV reporter systems which use either (1) HIV Gag proteins (e.g. vector pBR37R (Ludwig et al., 1999, J. Virol. 73:8279–8289)) or (2) a heterologous protein (e.g. chloramphenicol acetyl transferase=CAT; pDM128, Hope T. J. et al., 1990) as a reporter for Rev activity. In both systems the Rev reporter protein is detected in an indirect manner, i.e. either by means of antibody-ELISA (Gag, CAT) or, in the case of pDM128, by a functional CAT test. Both reporter proteins are detected in lysates of transfected cells for which several thousands of transfected cells must be employed.
The main disadvantages of the “gag-ELISA” are the relatively high efforts with respect to time and materials, the requirement of cell lysis (no further use of the cell population is possible), and the high costs (the techniques used so far are based on detecting Rev activity by the so-called “batch” method (antigen detection in the extract of lysed cells)).
So far, there have also been approaches which only detect the above-mentioned HIV Tat protein. Reporter gene constructs have been employed for this detection which for example express green fluorescent protein under the control of HIV LTR and thus in a Tat-dependent (but Rev-independent) manner (see Dorsky et al., J Acquir Immune Defic Syndr Hum Retrovirol, 1996 Dec. 1; 13(4):308–313 Detection of HIV-I infection with a green fluorescent protein reporter system).
Therefore, with regard to the disadvantages cited above the object of the present invention is to provide a highly specific reporter gene construct which may be used to perform HIV Rev-and Tat-specific test assays using living cells on a single cell level in a quick and cost-effective manner.
This object has been achieved by the features indicated in the independent claims. Preferred embodiments of the invention are set forth in the dependent claims.